Equipment
and
supplies

Reagents
and
solutions

Procedure:
Lung
tissue
preparation

Lungs
are
inflated
with
Feket's
acid
alcohol
formalin
and
allowed
to
fix
overnight.

Lungs
are
stored
in
70%
ethanol.

Lungs
are
trimmed
longitudinally
and
cross-sectionally
to
yield
a
minimum
of
4
lobes.

Tissues
are
embedded
in
paraffin
and
sectioned
at
6
microns.

Sections
are
stained
with
H&E.

Procedure:
Lung
histopathology

All
slides
are
reviewed
by
the
same
experienced
board-certified
pathologist
(JPS).

For
this
study,
diagnoses
are
limited
to
adenomas
and
occasional
primary
epithelial
hyperplasias
(argued
to
potentially
represent
an
early
stage
in
development
of
pulmonary
neoplasias).
Adenocarcinomas
are
not
included
in
this
study.

Lesions
are
defined
by
their
frequency
--
percentage
of
female
or
male
mice
per
strain
diagnosed
with
one
or
more
lesions.

Lesions
are
characterized
by
severity
based
on
tumor
size
and
numbers
per
mouse
(1=mild;
2=moderate;
3=severe;
4=extreme).

Data
are
recorded
and
coded
using
the
Mouse
Disease
Information
System
(MoDis;
Sundberg
et
al.,
2008;
Sundberg
et
al.,
2009).

Data
collected
by
investigator

Adenoma
frequency

Adenoma
severity
score
for
mice
with
tumors

MPD
computed
overall
strain
severity
score
(by
strain,
sex)
to
include
all
mice
tested
(mice
without
adenomas
were
considered
to
have
a
score
of
0).